Replaces Prod. #: ALX-200-603
Synthetically modified recombinant ubiquitin. The modification of the C-terminal glycine carboxyl into an aldehyde results in a potent, highly specific inhibitor of all ubiquitin deconjugating enzymes, including ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs) and deubiquitinylating enzymes (DUBs).
Product Details
| Alternative Name: | Ub-H |
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| Sequence: | Recombinant Ubiquitin (corresponding to UniProt sequence P62988). The C-terminal glycine carboxyl is synthetically modified to an aldehyde. |
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| MW: | 8.5kDa |
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| Source: | Produced in E.coli. |
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| UniProt ID: | P0CG47 (UBB), P0CG48 (UBC), P62979 (RPS27A), P62987 (UBA52) |
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| Formulation: | Liquid. In aqueous solution containing 0.15M HCl. |
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| Purity: | ≥95% (HPLC) |
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| Quality Control: | Ubiquitin Aldehyde (recombinant) activity was confirmed by its inhibition of human UCH-L1 and deubiquitinylation of Ubiquitin-AMC (Prod. No. BML-SE211). |
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| Activity: | Ubiquitin Aldehyde (recombinant) activity was confirmed by its inhibition of UCH-L3 (BML-UW9745) and USP2 (BML-UW9850) deubiquitinylation of Ubiquitin-AMC (BML-SE211). |
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| Specific Activity: | Ki=2.5nM vs. UCH isopeptidase-T. |
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| Application Notes: | Ubiquitin Aldehyde (recombinant) is useful in the stabilisation of ubiquitin-protein conjugates in vitro, enhancing their accumulation in cell lysates and tissue extracts. Inhibition of deubiquitinylating enzyme activity by Ubiquitin Aldehyde (recombinant) can be used to identify and confirm such activity and to determine the inhibition kinetics for a particular enzyme. Recommended concentration for maximal inhibition is 2-5µM.
Co-crystallisation of ubiquitin aldehyde with specific deubiquitinylating enzymes (the inhibitor mimics the natural ubiquitin substrate) has also been used to probe enzyme:substrate interactions. |
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| Shipping: | Blue Ice Not Frozen |
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| Long Term Storage: | +4°C |
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| Handling: | Do not neutralise until immediateley prior to use. Do not lyophilize. Avoid presence of amino-containing compounds. Soluble and stable in aqueous solution at pH <7.0. |
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| Technical Info/Product Notes: | Typical assay set-up:
Substrate concentration: 0.01-1.0µM.
Enzyme concentration, UCH-L1 (human) (recombinant) (His) (BML-UW9740): 10-100nM.
Inhibitor concentration: 0.01-1.0µM.
Release of AMC fluorescence by DUB enzymes can be monitored using 380nm excitation and 460nm emission wavelengths. |
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| Regulatory Status: | RUO - Research Use Only |
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Figure 1: Typical HPLC analysis of Ubiquitin Aldehyde (recombinant) (Prod. No. BML-UW8450). Method: Column: VYDAC 218TP54; A: 0.1% TFA/H2O; B: 0.1% TFA/CH3CN; Gradient: 25-45% B/20 mins. Monitoring: 230nm.
Figure 2: Typical DUB Assay.
Method: 12.5nM UCH-L1 (PGP9.5) (human) (recombinant) (His) (Prod. No.
BML-UW9740); 500nM Ubiquitin-AMC; 500nM Ubiquitin Aldehyde (recombinant); 50mM HEPES pH7.8, 0.5mM EDTA, 1mM DTT."
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Product Literature References
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Ubiquitin Carboxy-Terminal HydrolaseL3 Correlates with Human Sperm Count, Motility and Fertilization: M. Wang, et al.; PLoS One
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Pro-apoptotic F-box protein Fbxl7 regulates mitochondrial function by mediating the ubiquitylation and proteasomal degradation of survivin: Y. Liu, et al.; J. Biol. Chem.
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General Literature References
Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. : M. Hu et al.; EMBO J.
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The polyglutamine neurodegenerative protein ataxin-3 binds polyubiquitylated proteins and has ubiquitin protease activity. : B. Burnett et al.; Hum. Mol. Genet.
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A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. : A.Borodovsky et al.; EMBO J.
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Structural basis for the specificity of ubiquitin C-terminal hydrolases.: S. C. Johnston et al.; EMBO J.
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Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. : L. C. Dang et al.; Biochemistry
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Kinetic studies on the inhibition of isopeptidase T by ubiquitin aldehyde.: F. Melandri et al.; Biochemistry
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Recognition of modified forms of ribonuclease A by the ubiquitin system.: R. L. Dunten et al.; J. Biol. Chem.
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Ubiquitin-aldehyde: a general inhibitor of ubiquitin-recycling processes.: A. Hershko & I. A. Rose; PNAS
84, 1829 (1987),
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